The invention relates to compounds, methods and kits for the determination of FKBP-binding immunosuppressant drugs, such as sirolimus compounds and tacrolimus compounds, in samples, such as patient samples, known or suspected to contain such FKBP-binding immunosuppressant drugs.
The body relies upon a complex immune response system to distinguish self from non-self. At times, the body's immune system must be controlled in order to either augment a deficient response or suppress an excessive response. For example, when organs such as kidney, heart, heart-lung, bone marrow and liver are transplanted in humans, the body will often reject the transplanted tissue by a process referred to as allograft rejection.
In treating allograft rejection, the immune system is frequently suppressed in a controlled manner with drug therapy. Immunosuppressant drugs are carefully administered to transplant recipients in order to help prevent allograft rejection of non-self tissue. Two most commonly administered immunosuppressive drugs to prevent organ rejection in transplant patients are Cyclosporine (CSA) and FK-506 (FK or tacrolimus). Another drug that finds use as an immunosuppressant in the United States and other countries is sirolimus, also known as rapamycin. Derivatives of sirolimus are also said to be useful as immunosuppressants. Such derivatives include, for example, Everolimus.
The side effects associated with some immunosuppressant drugs can be controlled in part by carefully controlling the level of the drug present in a patient. Because the distribution and metabolism of immunosuppressant drugs can vary greatly between patients and because of the wide range and severity of adverse reactions, accurate monitoring of the drug level (therapeutic drug monitoring or TDM) is recommended for all patients receiving immunosuppressant drugs, especially pediatric patients and those patients with hepatic impairment. TDM is also recommended when potent inducers or inhibitors of the enzyme CYP3A4 are co-administered. In addition, if sirolimus or its derivative is concomitantly administered with cyclosporine, TDM is recommended because pharmacokinetics are altered during drug co-administration. For example, if sirolimus is administered concomitantly with cyclosporin rather than administered four hours apart, sirolimus trough levels increase. For this reason, as well as to limit certain side effects, TDM should allow for better clinical results in selected cases.
Sirolimus and tacrolimus are the most important immunosuppressant drugs for organ transplantation in humans. Therapeutic monitoring of concentrations of sirolimus, tacrolimus and other related immunosuppressant drugs in blood is required to optimize dosing regimens to ensure maximal immunosuppression with minimal toxicity. Although sirolimus and tacrolimus are highly effective immunosuppressive agents, their use must be carefully managed because the effective dose range is narrow and excessive dosage can result in serious side effects. On the other hand, too little dosage of sirolimus or tacrolimus can lead to tissue rejection.
Many whole blood assays for immunosuppressant drugs such as, for example, sirolimus compounds and tacrolimus compounds, require a step using reagents to extract (release or displace) the drug from blood constituents. The chemical structures of sirolimus and tacrolimus are shown in FIG. 1. These molecules, which have a portion of their respective chemical structures in common, bind to endogenous specific binding substances such as, for example, endogenous specific binding proteins, e.g., FK-binding proteins (FKBPs). The sirolimus or tacrolimus drug molecules and drug metabolite molecules, if necessary, should be dissociated from endogenous binding substances, particularly for conducting assays for these substances. It is important to have a releasing reagent to compete with the FKBP-binding immunosuppressant drugs for binding to the endogenous binding substances and thereby release the free FKBP-binding immunosuppressant drug molecules for detection in an assay. Release of free FKBP-binding immunosuppressant drug molecules results in better assay sensitivity. Sirolimus and FK-506 ester (FKE) have been used as releasing reagents for a tacrolimus assay to release tacrolimus compounds from endogenous binding proteins. However, FK-506 ester has exhibited cross-reactivity with antibodies used in tacrolimus assays. Furthermore, the use of sirolimus as a releasing reagent is problematic because of carry-over issues where both sirolimus and tacrolimus immunoassays are carried out in the same instrument side-by-side.
There is, therefore, a continuing need to develop fast, accurate and sensitive diagnostic methods to measure levels of FKBP-binding immunosuppressant drugs in patients. The methods should employ a releasing agent for the FKBP-binding immunosuppressant drugs that has minimal cross-reactivity with a specific binding member such as, for example, an antibody, for the FKBP-binding immunosuppressant drug that is employed in an assay for the drug.